De Novo Design of A Membrane‐Anchored Probe for Multidimensional Quantification of Endocytic Dynamics

Abstract

As a process of cellular uptake, endocytosis, with gradient acidity in different endocytic vesicles, is vital for the homeostasis of intracellular nutrients and other functions. To study the dynamics of endocytic pathway, a membrane-anchored pH probe, ECGreen, is synthesized to visualize endocytic vesicles under structured illumination microscopy (SIM), a super-resolution technology. Being sensitive to acidity with increasing fluorescence at low pH, ECGreen can differentiate early and late endosomes as well as endolysosomes. Meanwhile, membrane anchoring not only improves the durability of ECGreen, but also provides an excellent anti-photobleaching property for long-time imaging with SIM. Moreover, by taking these advantages of ECGreen, a multidimensional analysis model containing spatial, temporal, and pH information is successfully developed for elucidating the dynamics of endocytic vesicles and their interactions with mitochondria during autophagy, and reveals a fast conversion of endosomes near the plasma membrane.

Super-resolution analyzing spatial organization of lysosomes with an organic fluorescent probe

“…[13] To investigate the behavior of lysosomes during this process, we induced mitophagy, a form of autophagy that selectively degrades damaged mitochondria, by damaging mitochondria with carbonyl cyanide m -chlorophenyl hydrazone (CCCP), a common mitophagy inducer. [8b, 14] After treating HeLa cells with CCCP for 12 h, we observed that the shape of the mitochondria stained with MitoTracker Deep Red (MTDR) significantly changed and became shorter and more rounded (Figure 3A), and damaged mitochondria also lost their cristae in the inner membrane (Figures 3B and 3C).…”

“…Due to the strong connection between mitochondria and lysosomes, we exploited patient-derived SLC25A46 mutant fibroblasts; this led to highly fused mitochondria and decreased mitochondrial oxidative phosphorylation and resulted in clinical symptoms of mitochondrial diseases. [8g] This gene encodes a mitochondrial solute carrier protein family member, and its mutation results in neuropathy and optic atrophy. Even though the cell shapes of the SLC25A46 mutant fibroblasts were not different from the wild type (WT) (Figure S17), the mitochondria were relatively longer than those in the WT fibroblasts.…”

“…Due to its better resolution, super resolution imaging has been used extensively to investigate cellular contents. [8] Especially, the viscosity of mitochondria and lysosomes in live cells has been reported. [9] However, few studies have been dedicated to reveal the spatial organization between lysosomes and mitochondria using SIM techniques.…”

Light-activated mitochondrial fission through optogenetic control of mitochondria-lysosome contacts

“…Therefore, the induction of MLCs could potentially be used to accelerate mitochondrial fission. However, a method to directly regulate MLCs in a reversible and controllable manner in living cells is critical 29 – 32 .…”

Ultralong-Term Super-Resolution Tracking of Lysosomes in Brain Organoids by Near-Infrared Noble Metal Nanoclusters

“…Commercially available lysosomal dyes (e.g., LTR) are acidic pH-dependent and are repelled by lysosomes as their pH value increases. 27 Because nanoparticles enter lysosomes via endocytosis, the localization of the BSA−NCs was not expected to change with pH fluctuation. To gauge that difference between commercial dyes and the BSA−NCs, the stained cells were treated with chloroquine (100 μM, 30 min), which is a cell-permeable base, to increase the lysosomal pH.…”